Evidence-based Evaluation Of Glycyrrhizic Acid Preparations In The Prevention And Treatment Of Firstline Anti-tuberculosis Drug-induced Liver Injury And Its Mechanism Based On Gut Microbiota

Background:Tuberculosis remains a major public health problem in many countries,and although most patients benefit from a quadruple first-line anti-TB regimen of isoniazid,rifampicin,pyrazinamide,and ethambutol(HRZE),first-line anti-tuberculosis drug-induced liver injury(ATB-DILI)is the main cause of treatment interruption,and even severe liver failure and death in severe cases.Glycyrrhizic acid(GA)preparations are widely used anti-inflammatory and hepatoprotective drugs,and many clinical studies have found that they have good prevention and treatment effects on ATB-DILI,but it remains unclear as to which GA preparations is superior to the others.The mechanism of GA preparations against ATB-DILI also needs to be further elucidated.More and more studies have found that gut microbiota may be a potential pathogenic factor of DILI,gut microbiota and intestinal barrier function disruption can directly or indirectly contribute to the development of liver injury,while probiotic therapy plays an active role in various liver diseases.Studies have found that GA preparations have the function of improving gut microbiota imbalance and promoting intestinal barrier integrity,so the protective mechanism of glycyrrhizic acid preparations on ATB-DILI can be elucidated from the perspective of gut microbiota.Objectives:To comprehensively compare the prevention and treatment effects of different GA preparations on ATB-DILI using network meta-analysis,and establish a mouse ATB-DILI model,and the most effective glycyrrhizic acid preparation was selected to study and verify its protective mechanism on ATB-DILI from the perspective of gut microbiota and intestinal barrier.Methods:(1)A network meta-analysis(NMA)comparing the effectiveness of different glycyrrhizic acid preparations in the prevention and treatment of ATB-DILI:formulate a search strategy to conduct literature search,screen the literature according to the established inclusion and exclusion criteria,extract relevant information of the included literature.The NMA was conducted with a random-effects model under the Bayesian framework to calculate risk ratios(RRs)with 95%credible intervals(95%Cr Is)using R software(version 3.6.1).(2)To explore the protective mechanism of magnesium isoglycyrrhizinate(Mg IG)on ATB-DILI based on gut microbiota and intestinal barrier:Male BALB/c mice aged 6-8 weeks were selected and randomly divided into Control group(C group),magnesium isoglycyrrhizinate group(Mg group),anti-tuberculosis drug group(H group)and anti-tuberculosis drug combined with magnesium isoglycyrrhizinate group(HMg group),6 mice in each group.H group and HMg group were gavaged with HRZE mixed solution,the daily dose was77mg/kg rifampicin+39mg/kg isoniazid+195mg/kg pyrazinamide+156mg/kg ethambutol,2 hours later,Mg group and the HMg group was injected intraperitoneally with magnesium isoglycyrrhizinate at a daily dose of 40 mg/kg.The experimental period was five weeks.Mouse serum was collected to detect serum liver function,and intestinal permeability and lipopolysaccharide(LPS),feces were collected for 16S r DNA sequencing and analysis,and liver tissue was collected for pathology detection,oxidative stress levels,m RNA levels of inflammatory factors and inflammatory pathways detection,and colon tissue was collected for pathological detection and tight junction protein expression detection.(3)To verify whether supplementation with Lactobacillus rhamnosus JYLR-005 can alleviate ATB-DILI:the experiment was divided into control group(C group),tuberculosis drug group(H group),and anti-tuberculosis drug combined with Lactobacillus rhamnosus JYLR-005group(HLR group).The mice in C group and H group were treated the same as above,and the HLR group was gavaged with HRZE mixed solution,then 2 hours later,gavaged with Lactobacillus rhamnosus JYLR-005 aqueous solution(>10~9CFU/day/mice).The experimental period was five weeks.Except for mouse feces,other samples were collected and processed as above.Results:(1)In terms of preventing anti-TB DILI(33 RCTs,comprising 5,762patients),compound glycyrrhizin capsules or tablets(CGC),diammonium glycyrrhizinate capsules(DGC),diammonium glycyrrhizinate enteric capsules(DGEC),and diammonium glycyrrhizinate injection(DGI),but not compound glycyrrhizin injection(CGI),significantly reduced the incidence of liver injury than control group(RRs ranged from 0.26 to 0.58);CGC(RR:0.50,95%Cr I:0.27-0.94)and DGEC(RR:0.58,95%Cr I:0.35-0.96)were superior to DGC(RRs=0.50 and 0.58,respectively).In terms of treating anti-TB DILI(64 RCTs,comprising 5,161 patients),Mg IG was most effective among all regimens(RRs ranged from 1.15 to 1.72)while DGC ranked last(RRs ranged from 0.58 to 0.83).(2)Mg IG significantly reversed HRZE-increased serum liver function alanine aminotransferase(ALT),aspartate aminotransferase(AST)and alkaline phosphatase(AKP)levels,liver tissue malondialdehyde(MDA)levels and liver tissue pathological damage,and alleviate liver injury in mice.In addition,HRZE significantly down-regulated the abundance of Lactobacillus in mouse feces while Mg IG significantly enriched the abundance of Lactobacillus and other beneficial bacteria.In addition,Mg IG significantly up-regulated HRZE-induced low expression of tight junction protein 1(ZO-1)and occludin proteins in colon,significantly decreased intestinal permeability and serum LPS concentration,and inhibited HRZE-induced liver tissue high m RNA expression of Toll like receptor 2(TLR2),Toll like receptor 4(TLR4),nuclear factor-κB(NF-κB),tumor necrosis factorα(TNF-α),interleukin 1β(IL-1β)and interleukin 6(IL-6).(3)Supplementation of Lactobacillus rhamnosus JYLR-005significantly reversed HRZE-induced abnormal levels of ALT,AST and superoxide dismutase(SOD),up-regulate colonic occludin protein expression,reduce intestinal permeability and LPS concentration,and significantly inhibited HRZE-increased liver tissue m RNA levels of TLR2,TLR4,NF-κB,TNF-α,IL-1βand IL-6.Conclusions:(1)All GA preparations except for CGI were effective in preventing the incidence of ATB-DILI,and both CGC and DGEC were superior to DGC.Mg IG seems to be the best choice whereas DGC seems to be the worst choice among all GA preparations for the treatment of ATB-DILI.(2)Mg IG effectively improved HRZE-induced liver injury in mice and reduce oxidative stress and inflammation.Its protective mechanism may be related to enriching beneficial bacteria,enhancing intestinal barrier function and inhibiting LPS/TLRs/NF-κB signaling pathway activation.(3)Lactobacillus rhamnosus JYLR-005 supplementation can alleviate HRZE-induced liver injury to a certain extent,and can enhance intestinal barrier function and inhibit LPS/TLRs/NF-κB pathway to reduce HRZE-induced liver inflammation in mice.